Abstract: Hyper-activation of the Akt kinase and oncogenic function is a hallmark of breast cancer and has been shown to be tightly associated with chemotherapy and hormone therapy resistance, leading to a worse clinical outcome in breast cancer patients. Although substantial progress has been made in the past decades to understand the molecular mechanisms of Akt activation, our knowledge about the upstream signaling pathways controlling Akt kinase activity is far from complete that restrain us from targeting the Akt oncogenic signaling pathway for treating breast cancers. Accumulating evidence has shown that Akt can be activated by DNA damage or in Rb-deficient cells. However, the molecular mechanisms underlying Akt activation in either DNA damage or Rb-deficient conditions are largely unknown. The goal of this proposal is to uncover the molecular mechanisms underlying how the upstream signaling pathways, such as DNA-PK and Rb, regulate Akt to confer chemotherapy resistance in breast cancer, as well as to examine whether Akt inhibitors could synergize with DNA damage drugs or CDK4/6 inhibitors to suppress Akt signaling and overcome drugs resistance. To this end, in Aim 1, our preliminary data showed that during DNA damage response, DNA-PK- mediated Sin1 phosphorylation is required for DNA damage-induced Akt activation. We will further define the molecular mechanisms through which Sin1 phosphorylation by DNA-PK governs Akt activation under DNA damage condition. We will also determine whether synergistic usage of DNA damaging drugs and Akt inhibitors could more efficiently suppress breast cancer in both cell culture and mouse model systems. In Aim 2, we have obtained preliminary evidence to demonstrate that the Rb tumor suppressor inhibits Akt kinase activity largely through directly binding Sin1. Although Rb exerts its tumor suppressor role through suppressing E2F family of transcriptional factors, we will investigate the molecular mechanisms underlying the non- canonical tumor suppressive role of Rb in inhibiting Sin1 (mTORC2)-mediated activation of Akt. Moreover, we will examine whether inhibiting Rb phosphorylation would activate Akt to confer therapeutic drugs resistance, and determine whether Akt inhibitors would sensitize breast cancer cells to CDK4/6 inhibitors in vitro and in vivo. Therefore, the proposed studies will not only define a novel Sin1-dependent Akt activation mechanism, but also provide the rationale and molecular basis for the synergistic usage of chemotherapeutic drugs or CDK4/6 with Akt inhibitors clinically to overcome drugs resistance. My research career goals are to apply the biochemistry, cellular culture and mouse models to understand how the aberrant cancer signaling pathways contribute to breast cancer development and relapse, and to explore possible drug targets to achieve better clinical outcomes for breast cancer therapy. This K99/R00 award will allow me to improve my ability to design and execute experiments as well as solve problems. Moreover, this award will also facilitate my research career development through independently conducting scientific research and training future young scientists.